Anti-epidermal growth factor receptor variant III chimeric antigen receptors and use of same for the treatment of cancer

ABSTRACT

The invention provides chimeric antigen receptors (CARs) comprising an antigen binding domain of human antibody 139, an extracellular hinge domain, a transmembrane domain, and an intracellular domain T cell receptor signaling domain. Nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies, or antigen binding portions thereof, and pharmaceutical compositions relating to the CARs are disclosed. Methods of detecting the presence of cancer in a host and methods of treating or preventing cancer in a host are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.16/751,643, filed Jan. 24, 2020, which is a continuation of U.S.application Ser. No. 15/448,707, filed Mar. 3, 2017, now U.S. Pat. No.10,570,214, which is a divisional of U.S. application Ser. No.14/994,403, filed Jan. 13, 2016, now U.S. Pat. No. 9,624,306, which is adivisional of U.S. application Ser. No. 14/110,189, filed Oct. 24, 2013,now U.S. Pat. No. 9,266,960, which is the U.S. national phase ofInternational Patent Application No. PCT/US2012/029861, filed Mar. 21,2012, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/473,409, filed Apr. 8, 2011, each of which is incorporated byreference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under project numberZIABC010984 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 24,644 Byte ASCII (Text) file named“757120_ST25.TXT,” dated Aug. 13, 2021.

BACKGROUND OF THE INVENTION

The American Cancer Society estimates that approximately 20,500 newcases of primary brain and nervous system tumors will develop andapproximately 12,740 patients will die in the U.S. each year (Jemal etal., Cancer J. Clin., 57:43-66 (2007)) as a result of these cancers.Brain tumors account for approximately 85 to 90% of all central nervoussystem malignancies. Glioblastoma is the most aggressive and most commonglioma accounting for 51% of all gliomas (CBTRUS 2008 StatisticalReport: Primary Brain Tumors in the United States-CBTRUS, 2000-2004(2008)). Despite advances in conventional treatments such as surgicalresection, radiation therapy, and chemotherapy, the prognosis forgliomas, as well as other types of brain and nervous system cancer, maybe poor. For example, most patients with glioblastoma multiforme (GBM)survive less than 15 months from diagnosis. Accordingly, there exists anunmet need for additional treatments for cancer, particularly gliomas.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides chimeric antigen receptors(CARs) comprising an antigen binding domain of human antibody 139, anextracellular hinge domain, a transmembrane domain, and an intracellularT cell signaling domain.

Further embodiments of the invention provide related nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, and pharmaceuticalcompositions relating to the CARs of the invention.

Additional embodiments of the invention provide methods of detecting thepresence of cancer in a host and methods of treating or preventingcancer in a host.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1A is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled parent U87 glioblastoma tumor cell line (“U87”) (target cell) byhuman peripheral blood lymphocytes (PBL) (effector cells) that wereuntransduced (UnTd) (▪) or transduced with vectors encoding greenfluorescent protein (GFP) (●), SEQ ID NO: 10 (h139Ab-hCD828BBZ CAR) (x),or SEQ ID NO: 11 (h139Ab-hCD28Z) (▴) at various effector to targetratios (E:T ratio).

FIG. 1B is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled U87-GFP (expressing GFP) glioblastoma tumor cell line (targetcell) by human PBL (effector cells) that were untransduced (UnTd) (▪) ortransduced with vectors encoding GFP (●), SEQ ID NO: 10(h139Ab-hCD828BBZ CAR) (x), or SEQ ID NO: 11 (h139Ab-hCD28Z) (▴) atvarious effector to target ratios (E:T ratio).

FIG. 1C is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled U87-EGFR glioblastoma tumor cell line (expressing wild-typeepidermal growth factor receptor) (target cell) by human PBL (effectorcells) that were untransduced (UnTd) (▪) or transduced with vectorsencoding GFP (●), SEQ ID NO: 10 (h139Ab-hCD828BBZ CAR) (x), or SEQ IDNO: 11 (h139Ab-hCD28Z) (▴) at various effector to target ratios (E:Tratio).

FIG. 1D is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled U87-vIII glioblastoma tumor cell line (expressing EGFRvIII)(target cell) by human PBL (effector cells) that were untransduced(UnTd) (▪) or transduced with vectors encoding GFP (●), SEQ ID NO: 10(h139Ab-hCD828BBZ CAR) (x), or SEQ ID NO: 11 (h139Ab-hCD28Z) (▴) atvarious effector to target ratios (E:T ratio).

FIG. 2A is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled parent U251 glioblastoma tumor cell line (target cell) by humanPBL (effector cells) that were untransduced (UnTd) (▪) or transducedwith vectors encoding green fluorescent protein (GFP) (●), anti-ERBB2CAR (♦), SEQ ID NO: 10 (h139Ab-hCD828BBZ CAR) (x), or SEQ ID NO: 11(h139Ab-hCD28Z) (▴) at various effector to target ratios (E:T ratio).

FIG. 2B is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled U251-GFP glioblastoma tumor cell line (expressing GFP) (targetcell) by human PBL (effector cells) that were untransduced (UnTd) (▪) ortransduced with vectors encoding green fluorescent protein (GFP) (●),anti-ERBB2 CAR (♦), SEQ ID NO: 10 (h139Ab-hCD828BBZ CAR) (x), or SEQ IDNO: 11 (h139Ab-hCD28Z) (▴) at various effector to target ratios (E:Tratio).

FIG. 2C is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled U251-EGFR glioblastoma tumor cell line (expressing wild-typeEGFR) (target cell) by human PBL (effector cells) that were untransduced(UnTd) (▪) or transduced with vectors encoding green fluorescent protein(GFP) (●), anti-ERBB2 CAR (♦), SEQ ID NO: 10 (h139Ab-hCD828BBZ CAR) (x),or SEQ ID NO: 11 (h139Ab-hCD28Z) (▴) at various effector to targetratios (E:T ratio).

FIG. 2D is a graph showing the specific lysis (percent lysis) of ⁵¹Crlabeled U251-vIII glioblastoma tumor cell line (expressing wild-typeEGFRvIII) (target cell) by human PBL (effector cells) that wereuntransduced (UnTd) (▪) or transduced with vectors encoding greenfluorescent protein (GFP) (●), anti-ERBB2 CAR (♦), SEQ ID NO: 10(h139Ab-hCD828BBZ CAR) (x), or SEQ ID NO: 11 (h139Ab-hCD28Z) (▴) atvarious effector to target ratios (E:T ratio).

FIGS. 3A-3C are graphs showing interferon (IFN)-γ secretion as measuredby ELISA (pg/ml, mean of triplicate determinations) by untransducedcells (UT) or human T cells transduced with 3C10 CAR (FIG. 3A), L8A4 CAR(FIG. 3B), h139Ab-hCD28Z CAR (FIG. 3C) upon co-culture with target cellslines untransduced NIH-3T3 (3T3) (3T3 UT grey bars), untransduced BHK(BHK UT, dotted bars), untransduced 293GP (293GP UT, checkered bars),EGFR-wild type transduced 3T3 (3T3 EGFRwt, white bars), EGFR-wild typetransduced BHK (BHK EGFRwt, striped bars), EGFRvIII-transduced 3T3 (3T3EGFRvIII, black bars), EGFRvIII-transduced BHK (BHK EGFRvIII,cross-hatched bars), or EGFRvIII-transduced 293GP (293GP EGFRvIII, barswith vertical and horizontal stripes).

FIGS. 4A and 4B are graphs showing IFN-γ secretion as measured by ELISA(pg/ml, mean of triplicate determinations) by T-cells from Donor 2 (FIG.4A) or Donor 3 (FIG. 4B) that were transduced with green-fluorescentprotein (GFP) or the 139-28Z vector (unsorted) (139bulk), or T cellsthat were transduced with the h139Ab-hCD28Z vector and then bead sortedinto CD8 and CD4 enriched (>96%+) T cell populations (139CD8+ and139CD4+). IFN-γ was measured upon co-culture of the transduced cellsovernight with BHK target cells (black bars), EGFR wild type engineeredBHK cells (EGFRwt, grey bars) or EGFRvIII engineered BHK cells(EGFRvIII, cross-hatched bars) or media (checkered bars).

FIGS. 5A and 5B are graphs showing IFN-γ secretion by T cells from humanDonor 4 (FIG. 5A) and human Donor 5 (FIG. 5B) that were untransduced(UT) or transduced with anti-EGFRvIII CAR vector (139-28BBZ) (EGFRvIII),a GFP expressing vector (GFP), or a CAR vector targeting ERBB2.Transduced T cells were co-cultured with media (black bars), wild typeEGFR engineered U251 cells (U251-EGFRwt, white bars), EGFR variant IIIengineered U251 cells (U251-EGFRvIII, grey bars), or glioma stem celllines 1228 (bars with horizontal), 308 (dotted bars), or 822(cross-hatched bars).

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides chimeric antigen receptors(CARs) comprising an antigen binding domain of human antibody 139(h139Ab), an extracellular hinge domain, a transmembrane domain, and anintracellular T cell signaling domain.

A chimeric antigen receptor (CAR) is an artificially constructed hybridprotein or polypeptide containing the antigen binding domains of anantibody (e.g., scFv) linked to T-cell signaling domains.Characteristics of CARs include their ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT cells expressing CARs the ability to recognize antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

The phrases “have antigen specificity” and “elicit antigen-specificresponse” as used herein means that the CAR can specifically bind to andimmunologically recognize an antigen, such that binding of the CAR tothe antigen elicits an immune response.

The CARs of the invention have antigen specificity for epidermal growthfactor receptor variant III (EGFRvIII). EGFRvIII is a variant of theepidermal growth factor receptor (EGFR), which is a transmembraneglycoprotein that is a member of the protein kinase superfamily.EGFRvIII is the most prevalent of several EGFR mutations found in humangliomas, and is expressed in about 30% to about 50% of glioblastomamultiforme (GBM) (also known as “glioblastoma”). The expression ofEGFRvIII results from intragene deletion rearrangements that eliminateEGFR exons 2-7, and cause the joining of exons 1 and 8 of the codingsequences. EGFRvIII is expressed by tumor cells of various cancers suchas, e.g., glioblastoma (including glioblastoma stem cells); breast,ovarian, and non-small cell lung carcinomas; head and neck squamous cellcarcinoma; medulloblastoma, colorectal cancer, prostate cancer, andbladder carcinoma. Without being bound to a particular theory ormechanism, it is believed that by eliciting an antigen-specific responseagainst EGFRvIII, the inventive CARs provide for one or more of thefollowing: targeting and destroying EGFRvIII-expressing tumor cells,reducing or eliminating tumors, facilitatating infiltration of immunecells to the tumor site, and enhancing/extending anti-tumor responses.Because EGFRvIII is not expressed in normal (i.e., non-cancerous)tissue, it is contemplated that the inventive CARs advantageouslysubstantially avoid targeting/destroying normal tissues and cells.

The invention provides a CAR comprising an antigen binding domain ofhuman antibody 139. Antibody 139 is a human, anti-EGFRvIII antibody.Antibody 139 specifically binds to EGFRvIII. Suitable human antibody 139sequences are disclosed in, for example, U.S. Pat. No. 7,628,986, whichis hereby incorporated by reference. In this regard, a preferredembodiment of the invention provides CARs comprising an antigen-bindingdomain comprising, consisting of, or consisting essentially of, a singlechain variable fragment (scFv) of human antibody 139.

Human antibody 139 comprises a light chain variable region and a heavychain variable region. The light chain variable region may comprise,consist of, or consist essentially of SEQ ID NO: 1. The heavy chainvariable region may comprise, consist, or consist essentially of SEQ IDNO: 2. Accordingly, in an embodiment of the invention, the antigenbinding domain comprises a light chain variable region comprising SEQ IDNO: 1 and/or a heavy chain variable region comprising SEQ ID NO: 2.

In an embodiment, the antigen binding domain comprises a linker peptide.The linker peptide may be positioned between the light chain variableregion and the heavy chain variable region. In this regard, the antigenbinding domain may comprise a linker peptide comprising, consisting of,or consisting essentially of SEQ ID NO: 3.

In an embodiment, the antigen binding domain comprises a leadersequence. The leader sequence may be positioned at the amino terminus ofthe light chain variable region. In this regard, the antigen bindingdomain may comprise a leader sequence comprising, consisting of, orconsisting essentially of SEQ ID NO: 4.

In an embodiment, the antigen binding domain may comprise a leadersequence, a light chain variable region, a linker peptide, and a heavychain variable region. In this regard, the antigen binding domaincomprising a leader sequence, a light chain variable region, a linkerpeptide, and a heavy chain variable region comprises, consists of, orconsists essentially of SEQ ID NO: 5 (scFv human antibody 139).

In an embodiment of the invention, the CAR comprises an extracellularhinge domain, a transmembrane domain, and optionally, an intracellularhinge domain comprising CD8 and an intracellular T cell signaling domaincomprising CD28, 4-1BB, and CD3ζ. CD28 is a T cell marker important in Tcell co-stimulation. CD8 is also a T cell marker. 4-1BB transmits apotent costimulatory signal to T cells, promoting differentiation andenhancing long-term survival of T lymphocytes. CD3ζ associates with TCRsto produce a signal and contains immunoreceptor tyrosine-basedactivation motifs (ITAMs). In this regard, a preferred embodiment of theinvention provides an extracellular hinge domain and transmembranedomain comprising, consisting essentially of, or consisting of, SEQ IDNO: 6 (human CD8 extracellular hinge domain and transmembrane domain).The intracellular T cell signaling domain comprises, consistsessentially of, or consists of, SEQ ID NO: 7 (human CD28, 4-1BB, andCD3ζ intracellular T cell signaling domains).

In another embodiment of the invention, the CAR comprises anextracellular hinge domain, transmembrane domain, and intracellular Tcell signaling domain comprising CD28 and CD3ζ. In this regard, apreferred embodiment of the invention provides an extracellular hingedomain, transmembrane domain, and intracellular T cell signaling domaincomprising, consisting essentially of, or consisting of, SEQ ID NO: 8(human CD28 extracellular hinge, transmembrane domain, and intracellularT cell signaling domains) and SEQ ID NO: 9 (human CD3ζ intracellular Tcell signaling domain).

Additional embodiments of the invention provide CARs comprising,consisting of, or consisting essentially of any of the amino acidsequences set forth in Table 1.

TABLE 1 Sequence scFv Further Components SEQ ID NO: 10 human Human CD8extracellular hinge and (h139Ab- antibody 139 transmembrane domainshCD828BBZ) Human CD28, human 4-1BB, and human CD3ζ intracellular T cellsignaling domains SEQ ID NO: 11 human Human CD28 extracellular hinge and(h139Ab- antibody 139 transmembrane domains hCD28Z) Human CD28 and humanCD3ζ intracellular T cell signaling domains

The invention also provides related nucleic acids, recombinantexpression vectors, host cells, populations of cells, antibodies, orantigen binding portions thereof, and pharmaceutical compositionsrelating to the CARs of the invention.

Included in the scope of the invention are functional portions of theinventive CARs described herein. The term “functional portion” when usedin reference to a CAR refers to any part or fragment of the CAR of theinvention, which part or fragment retains the biological activity of theCAR of which it is a part (the parent CAR). Functional portionsencompass, for example, those parts of a CAR that retain the ability torecognize target cells, or detect, treat, or prevent a disease, to asimilar extent, the same extent, or to a higher extent, as the parentCAR. In reference to the parent CAR, the functional portion cancomprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, ormore, of the parent CAR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent CAR.Desirably, the additional amino acids do not interfere with thebiological function of the functional portion, e.g., recognize targetcells, detect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity, as compared tothe biological activity of the parent CAR.

Included in the scope of the invention are functional variants of theinventive CARs described herein. The term “functional variant” as usedherein refers to a CAR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent CAR, whichfunctional variant retains the biological activity of the CAR of whichit is a variant. Functional variants encompass, for example, thosevariants of the CAR described herein (the parent CAR) that retain theability to recognize target cells to a similar extent, the same extent,or to a higher extent, as the parent CAR. In reference to the parentCAR, the functional variant can, for instance, be at least about 30%,50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to theparent CAR.

A functional variant can, for example, comprise the amino acid sequenceof the parent CAR with at least one conservative amino acidsubstitution. Alternatively or additionally, the functional variants cancomprise the amino acid sequence of the parent CAR with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parent CAR.

Amino acid substitutions of the inventive CARs are preferablyconservative amino acid substitutions. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the same orsimilar chemical or physical properties. For instance, the conservativeamino acid substitution can be an acidic/negatively charged polar aminoacid substituted for another acidic/negatively charged polar amino acid(e.g., Asp or Glu), an amino acid with a nonpolar side chain substitutedfor another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val,Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positivelycharged polar amino acid substituted for another basic/positivelycharged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged aminoacid with a polar side chain substituted for another uncharged aminoacid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), anamino acid with a beta-branched side-chain substituted for another aminoacid with a beta-branched side-chain (e.g., Ile, Thr, and Val), an aminoacid with an aromatic side-chain substituted for another amino acid withan aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

The CAR can consist essentially of the specified amino acid sequence orsequences described herein, such that other components, e.g., otheramino acids, do not materially change the biological activity of thefunctional variant.

The CARs of embodiments of the invention (including functional portionsand functional variants) can be of any length, i.e., can comprise anynumber of amino acids, provided that the CARs (or functional portions orfunctional variants thereof) retain their biological activity, e.g., theability to specifically bind to antigen, detect diseased cells in ahost, or treat or prevent disease in a host, etc. For example, the CARcan be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100,125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or moreamino acids in length.

The CARs of embodiments of the invention (including functional portionsand functional variants of the invention) can comprise synthetic aminoacids in place of one or more naturally-occurring amino acids. Suchsynthetic amino acids are known in the art, and include, for example,aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid,homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The CARs of embodiments of the invention (including functional portionsand functional variants) can be glycosylated, amidated, carboxylated,phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfidebridge, or converted into an acid addition salt and/or optionallydimerized or polymerized, or conjugated.

The CARs of embodiments of the invention (including functional portionsand functional variants thereof) can be obtained by methods known in theart. The CARs may be made by any suitable method of making polypeptidesor proteins. Suitable methods of de novo synthesizing polypeptides andproteins are described in references, such as Chan et al., Fmoc SolidPhase Peptide Synthesis, Oxford University Press, Oxford, UnitedKingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., MarcelDekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., OxfordUniversity Press, Oxford, United Kingdom, 2001; and U.S. Pat. No.5,449,752. Also, polypeptides and proteins can be recombinantly producedusing the nucleic acids described herein using standard recombinantmethods. See, for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, Cold SpringHarbor, NY 2001; and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.Further, some of the CARs of the invention (including functionalportions and functional variants thereof) can be isolated and/orpurified from a source, such as a plant, a bacterium, an insect, amammal, e.g., a rat, a human, etc. Methods of isolation and purificationare well-known in the art. Alternatively, the CARs described herein(including functional portions and functional variants thereof) can becommercially synthesized by companies, such as Synpep (Dublin, CA),Peptide Technologies Corp. (Gaithersburg, MD), and Multiple PeptideSystems (San Diego, CA). In this respect, the inventive CARs can besynthetic, recombinant, isolated, and/or purified.

An embodiment of the invention further provides an antibody, or antigenbinding portion thereof, which specifically binds to an epitope of theCARs of the invention. The antibody can be any type of immunoglobulinthat is known in the art. For instance, the antibody can be of anyisotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can bemonoclonal or polyclonal. The antibody can be a naturally-occurringantibody, e.g., an antibody isolated and/or purified from a mammal,e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. The antibody can bein monomeric or polymeric form. Also, the antibody can have any level ofaffinity or avidity for the functional portion of the inventive CAR.

Methods of testing antibodies for the ability to bind to any functionalportion of the inventive CAR are known in the art and include anyantibody-antigen binding assay, such as, for example, radioimmunoassay(RIA), ELISA, Western blot, immunoprecipitation, and competitiveinhibition assays (see, e.g., Janeway et al., infra, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing, New York, NY(2001)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266 A1).

Phage display furthermore can be used to generate an antibody. In thisregard, phage libraries encoding antigen-binding variable (V) domains ofantibodies can be generated using standard molecular biology andrecombinant DNA techniques (see, e.g., Sambrook et al., supra, andAusubel et al., supra). Phage encoding a variable region with thedesired specificity are selected for specific binding to the desiredantigen, and a complete or partial antibody is reconstituted comprisingthe selected variable domain. Nucleic acid sequences encoding thereconstituted antibody are introduced into a suitable cell line, such asa myeloma cell used for hybridoma production, such that antibodieshaving the characteristics of monoclonal antibodies are secreted by thecell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S.Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235, 959-973 (1994).

An embodiment of the invention also provides antigen binding portions ofany of the antibodies described herein. The antigen binding portion canbe any portion that has at least one antigen binding site, such as Fab,F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichis a truncated Fab fragment including the variable (V) domain of anantibody heavy chain linked to a V domain of a light antibody chain viaa synthetic peptide, can be generated using routine recombinant DNAtechnology techniques (see, e.g., Janeway et al., supra). Similarly,disulfide-stabilized variable region fragments (dsFv) can be prepared byrecombinant DNA technology (see, e.g., Reiter et al., ProteinEngineering, 7, 697-704 (1994)). Antibody fragments of the invention,however, are not limited to these exemplary types of antibody fragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

Further provided by an embodiment of the invention is a nucleic acidcomprising a nucleotide sequence encoding any of the CARs describedherein (including functional portions and functional variants thereof).An embodiment of the invention provides a nucleic acid comprising anucleotide sequence encoding an antigen binding domain of human antibody139 comprising SEQ ID NO: 12 (encoding the leader sequence, light chainvariable region of human antibody 139, linker peptide, and heavy chainvariable region of human antibody 139). In this regard, an embodiment ofthe invention provides nucleic acids comprising, consisting of, orconsisting essentially of the nucleotide sequences of Table 2:

TABLE 2 Sequence scFv Further Components SEQ ID NO: human Human CD8extracellular hinge and 13 (h139Ab- antibody 139 transmembrane domainshCD828BBZ) Human CD28, human 4-1BB, and human CD3ζ intracellular T cellsignaling domains SEQ ID NO: human Human CD28 extracellular hinge and 14(h139Ab- antibody 139 transmembrane domains hCD28Z) Human CD28 and humanCD3ζ intracellular T cell signaling domains

“Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In some embodiments, the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

The nucleic acids of an embodiment of the invention may be recombinant.As used herein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

A recombinant nucleic acid may be one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques, such as thosedescribed in Sambrook et al., supra. The nucleic acids can beconstructed based on chemical synthesis and/or enzymatic ligationreactions using procedures known in the art. See, for example, Sambrooket al., supra, and Ausubel et al., supra. For example, a nucleic acidcan be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed upon hybridization (e.g., phosphorothioate derivatives andacridine substituted nucleotides). Examples of modified nucleotides thatcan be used to generate the nucleic acids include, but are not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Col.) and Synthegen (Houston,TX).

The nucleic acid can comprise any isolated or purified nucleotidesequence which encodes any of the CARs or functional portions orfunctional variants thereof. Alternatively, the nucleotide sequence cancomprise a nucleotide sequence which is degenerate to any of thesequences or a combination of degenerate sequences.

An embodiment of the invention also provides an isolated or purifiednucleic acid comprising a nucleotide sequence which is complementary tothe nucleotide sequence of any of the nucleic acids described herein ora nucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditions mayhybridize under high stringency conditions. By “high stringencyconditions” is meant that the nucleotide sequence specificallyhybridizes to a target sequence (the nucleotide sequence of any of thenucleic acids described herein) in an amount that is detectably strongerthan non-specific hybridization. High stringency conditions includeconditions which would distinguish a polynucleotide with an exactcomplementary sequence, or one containing only a few scatteredmismatches from a random sequence that happened to have a few smallregions (e.g., 3-10 bases) that matched the nucleotide sequence. Suchsmall regions of complementarily are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive CARs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

In an embodiment, the nucleic acids of the invention can be incorporatedinto a recombinant expression vector. In this regard, an embodiment ofthe invention provides recombinant expression vectors comprising any ofthe nucleic acids of the invention. For purposes herein, the term“recombinant expression vector” means a genetically-modifiedoligonucleotide or polynucleotide construct that permits the expressionof an mRNA, protein, polypeptide, or peptide by a host cell, when theconstruct comprises a nucleotide sequence encoding the mRNA, protein,polypeptide, or peptide, and the vector is contacted with the cell underconditions sufficient to have the mRNA, protein, polypeptide, or peptideexpressed within the cell. The vectors of the invention are notnaturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. The inventive recombinant expression vectors cancomprise any type of nucleotides, including, but not limited to DNA andRNA, which can be single-stranded or double-stranded, synthesized orobtained in part from natural sources, and which can contain natural,non-natural or altered nucleotides. The recombinant expression vectorscan comprise naturally-occurring or non-naturally-occurringinternucleotide linkages, or both types of linkages. Preferably, thenon-naturally occurring or altered nucleotides or internucleotidelinkages do not hinder the transcription or replication of the vector.

In an embodiment, the recombinant expression vector of the invention canbe any suitable recombinant expression vector, and can be used totransform or transfect any suitable host. Suitable vectors include thosedesigned for propagation and expansion or for expression or both, suchas plasmids and viruses. The vector can be selected from the groupconsisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD),the pBluescript series (Stratagene, LaJolla, CA), the pET series(Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophagevectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149,also can be used. Examples of plant expression vectors include pBI01,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animalexpression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Therecombinant expression vector may be a viral vector, e.g., a retroviralvector.

A number of transfection techniques are generally known in the art (see,e.g., Graham et al., Virology, 52: 456-467 (1973); Sambrook et al.,supra; Davis et al., Basic Methods in Molecular Biology, Elsevier(1986); and Chu et al., Gene, 13: 97 (1981). Transfection methodsinclude calcium phosphate co-precipitation (see, e.g., Graham et al.,supra), direct micro injection into cultured cells (see, e.g., Capecchi,Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al.,BioTechniques, 6: 742-751 (1988)), liposome mediated gene transfer (see,e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediatedtransduction (see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high velocitymicroprojectiles (see, e.g., Klein et al., Nature, 327: 70-73 (1987)).

In an embodiment, the recombinant expression vectors of the inventioncan be prepared using standard recombinant DNA techniques described in,for example, Sambrook et al., supra, and Ausubel et al., supra.Constructs of expression vectors, which are circular or linear, can beprepared to contain a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived, e.g., fromColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

The recombinant expression vector may comprise regulatory sequences,such as transcription and translation initiation and termination codons,which are specific to the type of host (e.g., bacterium, fungus, plant,or animal) into which the vector is to be introduced, as appropriate,and taking into consideration whether the vector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the CAR(including functional portions and functional variants thereof), or tothe nucleotide sequence which is complementary to or which hybridizes tothe nucleotide sequence encoding the CAR. The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, or a promoter found in the long-terminalrepeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Further, the recombinant expression vectors can be made to include asuicide gene. As used herein, the term “suicide gene” refers to a genethat causes the cell expressing the suicide gene to die. The suicidegene can be a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews, Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive CARs (including any ofthe functional portions or variants thereof), nucleic acids, recombinantexpression vectors, host cells, populations of host cells, orantibodies, or antigen binding portions thereof. Conjugates, as well asmethods of synthesizing conjugates in general, are known in the art(See, for instance, Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005)and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).

An embodiment of the invention further provides a host cell comprisingany of the recombinant expression vectors described herein. As usedherein, the term “host cell” refers to any type of cell that can containthe inventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5αE. coli cells,Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293cells, and the like. For purposes of amplifying or replicating therecombinant expression vector, the host cell may be a prokaryotic cell,e.g., a DH5α cell. For purposes of producing a recombinant CAR, the hostcell may be a mammalian cell. The host cell may be a human cell. Whilethe host cell can be of any cell type, can originate from any type oftissue, and can be of any developmental stage, the host cell may be aperipheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell(PBMC). The host cell may be a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. The T cell may be a human T cell. The T cell may be a Tcell isolated from a human. The T cell can be any type of T cell and canbe of any developmental stage, including but not limited to, CD4⁺/CD8⁺double positive T cells, CD4⁺ helper T cells, e.g., Th₁ and Th₂ cells,CD8⁺ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memoryT cells, naive T cells, and the like. The T cell may be a CD8⁺ T cell ora CD4⁺ T cell.

Also provided by an embodiment of the invention is a population of cellscomprising at least one host cell described herein. The population ofcells can be a heterogeneous population comprising the host cellcomprising any of the recombinant expression vectors described, inaddition to at least one other cell, e.g., a host cell (e.g., a T cell),which does not comprise any of the recombinant expression vectors, or acell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, anerythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, amuscle cell, a brain cell, etc. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly host cells (e.g., consisting essentially of) comprisingthe recombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

CARs (including functional portions and variants thereof), nucleicacids, recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive CAR materials”hereinafter, can be isolated and/or purified. The term “isolated” asused herein means having been removed from its natural environment. Theterm “purified” or “isolated” does not require absolute purity orisolation; rather, it is intended as a relative term. Thus, for example,a purified (or isolated) host cell preparation is one in which the hostcell is more pure than cells in their natural environment within thebody. Such host cells may be produced, for example, by standardpurification techniques. In some embodiments, a preparation of a hostcell is purified such that the host cell represents at least about 50%,for example at least about 70%, of the total cell content of thepreparation. For example, the purity can be at least about 50%, can begreater than about 60%, about 70% or about 80%, or can be about 100%.

The inventive CAR materials can be formulated into a composition, suchas a pharmaceutical composition. In this regard, an embodiment of theinvention provides a pharmaceutical composition comprising any of theCARs, functional portions, functional variants, nucleic acids,expression vectors, host cells (including populations thereof), andantibodies (including antigen binding portions thereof), and apharmaceutically acceptable carrier. The inventive pharmaceuticalcompositions containing any of the inventive CAR materials can comprisemore than one inventive CAR material, e.g., a CAR and a nucleic acid, ortwo or more different CARs. Alternatively, the pharmaceuticalcomposition can comprise an inventive CAR material in combination withother pharmaceutically active agents or drugs, such as chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In apreferred embodiment, the pharmaceutical composition comprises theinventive host cell or populations thereof.

The inventive CAR materials can be provided in the form of a salt, e.g.,a pharmaceutically acceptable salt. Suitable pharmaceutically acceptableacid addition salts include those derived from mineral acids, such ashydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, andsulphuric acids, and organic acids, such as tartaric, acetic, citric,malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, andarylsulphonic acids, for example, p-toluenesulphonic acid.

With respect to pharmaceutical compositions, the pharamaceuticallyacceptable carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the active agent(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive CAR material, as well as by the particular method used toadminister the inventive CAR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Preservatives may be used. Suitable preservatives mayinclude, for example, methylparaben, propylparaben, sodium benzoate, andbenzalkonium chloride. A mixture of two or more preservatives optionallymay be used. The preservative or mixtures thereof are typically presentin an amount of about 0.0001% to about 2% by weight of the totalcomposition.

Suitable buffering agents may include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. A mixture of two or more buffering agents optionally may beused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition.

The concentration of inventive CAR material in the pharmaceuticalformulations can vary, e.g., from less than about 1%, usually at or atleast about 10%, to as much as about 20% to about 50% or more by weight,and can be selected primarily by fluid volumes, and viscosities, inaccordance with the particular mode of administration selected.

Methods for preparing administrable (e.g., parenterally administrable)compositions are known or apparent to those skilled in the art and aredescribed in more detail in, for example, Remington: The Science andPractice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1,2005).

The following formulations for oral, aerosol, parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), and topical administration are merelyexemplary and are in no way limiting. More than one route can be used toadminister the inventive CAR materials, and in certain instances, aparticular route can provide a more immediate and more effectiveresponse than another route.

Formulations suitable for oral administration can comprise or consist of(a) liquid solutions, such as an effective amount of the inventive CARmaterial dissolved in diluents, such as water, saline, or orange juice;(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard orsoftshelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the inventive CAR material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the inventive CAR material in an inert base, such as gelatinand glycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

Formulations suitable for parenteral administration include aqueous andnonaqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive CAR material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof

The parenteral formulations will typically contain, for example, fromabout 0.5% to about 25% by weight of the inventive CAR material insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having, for example, ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range, forexample, from about 5% to about 15% by weight. Suitable surfactantsinclude polyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

Injectable formulations are in accordance with an embodiment of theinvention. The requirements for effective pharmaceutical carriers forinjectable compositions are well-known to those of ordinary skill in theart (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. LippincottCompany, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250(1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages622-630 (1986)).

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of embodiments of the invention for applicationto skin. The inventive CAR material, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

An “effective amount” or “an amount effective to treat” refers to a dosethat is adequate to prevent or treat cancer in an individual. Amountseffective for a therapeutic or prophylactic use will depend on, forexample, the stage and severity of the disease or disorder beingtreated, the age, weight, and general state of health of the patient,and the judgment of the prescribing physician. The size of the dose willalso be determined by the active selected, method of administration,timing and frequency of administration, the existence, nature, andextent of any adverse side-effects that might accompany theadministration of a particular active, and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousdiseases or disorders could require prolonged treatment involvingmultiple administrations, perhaps using the inventive CAR materials ineach or various rounds of administration. By way of example and notintending to limit the invention, the dose of the inventive CAR materialcan be about 0.001 to about 1000 mg/kg body weight of the subject beingtreated/day, from about 0.01 to about 10 mg/kg body weight/day, about0.01 mg to about 1 mg/kg body weight/day. When the inventive CARmaterial is a host cell, an exemplary dose of host cells may be aminimum of about one million cells (1 mg cells/dose). When the inventiveCAR material is a nucleic acid packaged in a virus, an exemplary dose ofvirus may be about 1 ng/dose.

For purposes of the invention, the amount or dose of the inventive CARmaterial administered should be sufficient to effect a therapeutic orprophylactic response in the subject or animal over a reasonable timeframe. For example, the dose of the inventive CAR material should besufficient to bind to antigen, or detect, treat or prevent disease in aperiod of from about 2 hours or longer, e.g., about 12 to about 24 ormore hours, from the time of administration. In certain embodiments, thetime period could be even longer. The dose will be determined by theefficacy of the particular inventive CAR material and the condition ofthe animal (e.g., human), as well as the body weight of the animal(e.g., human) to be treated.

For purposes of the invention, an assay, which comprises, for example,comparing the extent to which target cells are lysed and/or IFN-γ issecreted by T cells expressing the inventive CAR upon administration ofa given dose of such T cells to a mammal, among a set of mammals ofwhich is each given a different dose of the T cells, could be used todetermine a starting dose to be administered to a mammal. The extent towhich target cells are lysed and/or IFN-γ is secreted uponadministration of a certain dose can be assayed by methods known in theart.

In addition to the aforedescribed pharmaceutical compositions, theinventive CAR materials can be formulated as inclusion complexes, suchas cyclodextrin inclusion complexes, or liposomes. Liposomes can serveto target the inventive CAR materials to a particular tissue. Liposomesalso can be used to increase the half-life of the inventive CARmaterials. Many methods are available for preparing liposomes, asdescribed in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9,467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369.

The delivery systems useful in the context of embodiments of theinvention may include time-released, delayed release, and sustainedrelease delivery systems such that the delivery of the inventivecomposition occurs prior to, and with sufficient time to cause,sensitization of the site to be treated. The inventive composition canbe used in conjunction with other therapeutic agents or therapies. Suchsystems can avoid repeated administrations of the inventive composition,thereby increasing convenience to the subject and the physician, and maybe particularly suitable for certain composition embodiments of theinvention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-di-and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active composition is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

One of ordinary skill in the art will readily appreciate that theinventive CAR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive CAR materials is increased through the modification. Forinstance, the inventive CAR materials can be conjugated either directlyor indirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., inventive CAR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616.

Alternatively, the inventive CAR materials can be modified into a depotform, such that the manner in which the inventive CAR materials isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of inventive CAR materials can be, forexample, an implantable composition comprising the inventive CARmaterials and a porous or non-porous material, such as a polymer,wherein the inventive CAR materials are encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the inventive CAR materials are released from the implant at apredetermined rate.

When the inventive CAR materials are administered with one or moreadditional therapeutic agents, one or more additional therapeutic agentscan be coadministered to the mammal. By “coadministering” is meantadministering one or more additional therapeutic agents and theinventive CAR materials sufficiently close in time such that theinventive CAR materials can enhance the effect of one or more additionaltherapeutic agents, or vice versa. In this regard, the inventive CARmaterials can be administered first and the one or more additionaltherapeutic agents can be administered second, or vice versa.Alternatively, the inventive CAR materials and the one or moreadditional therapeutic agents can be administered simultaneously. Anexemplary therapeutic agent that can be co-administered with the CARmaterials is IL-2. It is believed that IL-2 enhances the therapeuticeffect of the inventive CAR materials. For purposes of the inventivemethods, wherein host cells or populations of cells are administered tothe host, the cells can be cells that are allogeneic or autologous tothe host.

It is contemplated that the inventive pharmaceutical compositions, CARs,nucleic acids, recombinant expression vectors, host cells, orpopulations of cells can be used in methods of treating or preventing adisease in a host. Without being bound to a particular theory ormechanism, the inventive CARs have biological activity, e.g., ability torecognize antigen, e.g., EGFRvIII, such that the CAR when expressed by acell is able to mediate an immune response against the cell expressingthe antigen, e.g., EGFRvIII, for which the CAR is specific. In thisregard, an embodiment of the invention provides a method of treating orpreventing cancer in a host, comprising administering to the host theCARs, the nucleic acids, the recombinant expression vectors, the hostcells, the population of cells, the antibodies and/or the antigenbinding portions thereof, and/or the pharmaceutical compositions of theinvention in an amount effective to treat or prevent cancer in the host.

An embodiment of the invention further comprises lymphodepleting thehost prior to administering the inventive CAR materials. Examples oflymphodepletion include, but may not be limited to, nonmyeloablativelymphodepleting chemotherapy, myeloablative lymphodepletingchemotherapy, total body irradiation, etc.

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the host. Preferably, the cells are autologous to thehost.

The host referred to herein can be any host. The host may be a mammal.As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. Themammals may be from the order Carnivora, including Felines (cats) andCanines (dogs). The mammals may be from the order Artiodactyla,including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). The mammals may be of theorder Primates, Ceboids, or Simoids (monkeys) or of the orderAnthropoids (humans and apes). Preferably, the mammal is a human.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma),bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancerof the anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, headand neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkinlymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia,liquid tumors, liver cancer, lung cancer (e.g., non-small cell lungcarcinoma), lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovariancancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer,pharynx cancer, prostate cancer, rectal cancer, renal cancer, skincancer, small intestine cancer, soft tissue cancer, solid tumors,stomach cancer, testicular cancer, thyroid cancer, and ureter cancer.Preferably, the cancer is glioma (e.g., ependymoma, astrocytoma,oligodendroglioma, and oligoastrocytoma), more preferably, glioblastomamultiforme (GBM) (also known as glioblastoma, astrocytoma grade IV, andgrade IV astrocytoma). Preferably, the cancer is characterized by theexpression of EGFRvIII.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof

Another embodiment of the invention provides a use of the inventiveCARs, nucleic acids, recombinant expression vectors, host cells,populations of cells, antibodies, or antigen binding portions thereof,or pharmaceutical compositions, for the treatment or prevention ofcancer in a host.

Another embodiment of the invention provides a method of detecting thepresence of cancer in a host, comprising: (a) contacting a samplecomprising one or more cells from the host with the CARs, the nucleicacids, the recombinant expression vectors, the host cells, thepopulation of cells, the antibodies, and/or the antigen binding portionsthereof of the invention, thereby forming a complex, (b) and detectingthe complex, wherein detection of the complex is indicative of thepresence of cancer in the host.

The sample may be obtained by any suitable method, e.g., biopsy ornecropsy. A biopsy is the removal of tissue and/or cells from anindividual. Such removal may be to collect tissue and/or cells from theindividual in order to perform experimentation on the removed tissueand/or cells. This experimentation may include experiments to determineif the individual has and/or is suffering from a certain condition ordisease-state. The condition or disease may be, e.g., cancer.

With respect to an embodiment of the inventive method of detecting thepresence of cancer in a host, the sample comprising cells of the hostcan be a sample comprising whole cells, lysates thereof, or a fractionof the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, awhole protein fraction, or a nucleic acid fraction. If the samplecomprises whole cells, the cells can be any cells of the host, e.g., thecells of any organ or tissue, including blood cells or endothelialcells.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the host. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs, polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells,populations of cells, or antibodies, or antigen binding portionsthereof, described herein, can be labeled with a detectable label suchas, for instance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

Methods of testing a CAR for the ability to recognize target cells andfor antigen specificity are known in the art. For instance, Clay et al.,J. Immunol., 163: 507-513 (1999), teaches methods of measuring therelease of cytokines (e.g., interferon-γ, granulocyte/monocyte colonystimulating factor (GM-CSF), tumor necrosis factor a (TNF-α) orinterleukin 2 (IL-2)). In addition, CAR function can be evaluated bymeasurement of cellular cytoxicity, as described in Zhao et al., J.Immunol., 174: 4415-4423 (2005).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates that a CAR comprising SEQ ID NO: 10(h139Ab-hCD828BBZ) or SEQ ID NO: 11 (h139Ab-hCD28Z) produces IFN-gammafollowing co-culture with EGFRvIII engineered target cell lines.

Chimeric antigen receptors targeting EGFRvIII were produced by combiningsingle chain antibody sequences from 7 different anti-EGFRvIIIantibodies to the T cell signaling domains of CD28 and CD3zeta. A totalof 9 different constructs were assembled (in 2 constructs the order ofthe VL and VH were switched) based on murine antibodies 3C10, MR-1, Y10,L8A4, and human antibodies 131, 139, and 13.1.2, which were insertedinto the γ-retroviral vector MSGV1. The expression of each construct wastested by transducing peripheral blood lymphocytes (PBL) andfluorescence-activated cell sorting (FACS) analysis using an anti-Fabspecific reagent (or protein L in later experiments). Three of the ninevectors constructed reproducible demonstrated CAR expression intransduced PBL, specifically those CARs based on antibodies 3C10, L8A4,and 139 (SEQ ID NO: 11) were shown to have cell surface staining intransduced PBL.

To test the biological activity of these 3 anti-EGFRvIII CAR constructs,γ-retroviral vector supernatant was produced and used to transduce PBL,which were co-cultured with EGFRvIII-expressing target cell lines. Inorder to develop an in vitro system to evaluate potential EGFRvIIItargeting vectors, an appropriate target cell line was establishedbecause no known glioblastoma cell lines express EGFRvIII. The wild typeEGFR gene was obtained from commercial sources and the vIII form wasconstructed by polymerase chain reaction (PCR) and inserted into aretroviral vector, which coexpressed a NeoR gene. Several cell lines(NIH-3T3, BHK, HEK-293GP, U87, and U251) were transduced and selectedand EGFRvIII expression was determined by a vIII specific antibody.

Specific IFN-gamma production was demonstrated for all three constructsby co-culture with EGFRvIII engineered established cell lines (FIGS. 3A,3B, and 3C, representative data for co-cultures with NIH-3T3, BHK, and293GP derived lines). BHK cells (BHK), EGFR transduced BHK (BHK-EGFRwt),EGFRvIII transduced BHK (BHK-EGFR vIII), 3T3 cells (3T3UT), EGFRtransduced 3T3 (3T3-EGFRwt), EGFRvIII transduced 3T3 (3T3-EGFRvIII),293GP cells (293GPUT), or EGFRvIII transduced 293GP (293GP-EGFRvIII),were co-cultured with the indicated CAR-transduced PBL (or untransduced(UT) PBL as controls) and IFN-gamma levels determined (values areIFN-gamma in pg/ml following overnight co-culture). In these co-cultureassays, all three CARs 3C10, L8A4 and 139 yielded specific IFN-γproduction when exposed to EGFRvIII expressing target cells, but notcells engineered to over express the wild type EGFR gene. Based on theobservation that the 139 CAR was slightly more reactive and is of humanorigin, and therefore less likely to be immunogenic in patients, allsubsequent assays were done with the 139 scFv-based CAR construct (SEQID NO: 11). T cells from two donors that were transduced with the139-CAR were sorted into CD8 and CD4 T cell populations andindependently tested for reactivity (FIGS. 4A (Donor 2) and 4B (Donor3)). Both CD4 and CD8 T cells specifically produced IFN-γ in co-culturewith EGFRvIII target cells.

The addition of T cell signaling elements from the 41BB co-stimulatorymolecule can enhance the survival of CAR engineered T cells. A newconstruct was assembled using signaling domains from CD28-41BB-CD3zeta(SEQ ID NO: 13) and compared to the original CD28-CD3zeta construct (SEQID NO: 14). While detection of the 28BBZ CAR vector construction by FACSwas less than the 28Z construct, the transduced T cells were equallyreactive against EGFRvIII expressing targets. T cells were transducedwith these vectors and control vectors (GFP or the Her2/neu CAR) andco-cultured with engineered glioblastoma cell lines and glioblastomamultiforme tumor stem cell (GBM-TSC) lines (Tables 3A and 3B).Established glioblastoma lines U87 and U251 were engineered to express acontrol GFP gene, the wild type EGFR gene (EGFRwt), or the EGFRvIII gene(EGFRvIII). These target cells or GBM-TSC lines 308, 822, and 1228 wereco-cultured with T cells transduced with the EGFRvIII-CAR vectorscontaining CD28-CD3zeta (139-28Z) (SEQ ID NO: 14) or CD28-41BB-CD3zeta(139-BBZ; h139Ab-hCD828BBZ) (SEQ ID NO: 13) signaling domains. IFN-γlevels were determined (values are IFN-γ in pg/ml following overnightco-culture with glioblastoma cell lines engineered to express EGFRvIII).Additional T cell controls included UT-untransduced PBL, GFP-GFP vectortransduced PBL, and a Her2/neu specific CAR. Biological activity, asdetermined by IFN-γ release (Table 3A and 3B), demonstrated that the twodifferent vector-transduced T cells were equally reactive againstEGFRvIII expressing glioma cell lines U87 and U251.

TABLE 3A Effector U87 Cells (IFN-γ, pg/ml) U251 Cells (IFN-γ, pg/ml)T-cell GFP EGFR wt EGFRvIII UT EGFR wt EGFRvIII GFP 389 236 339 0 0 0139-28Z 451 561 1797 0 0 2743 139-28BBZ 460 499 2117 0 0 1820 ERBB2 1061671 932 1195 2201 2692

TABLE 3B UT GFP 139-28Z 139-BBZ HER2/Neu GBM-TSC 308 0 35 987 1123 578GBM-TSC 822 0 95 1683 2267 372 GBM-TSC 1228 0 0 1387 1493 371

EXAMPLE 2

This example demonstrates that a CAR comprising SEQ ID NO: 10(h139Ab-hCD828BBZ) or SEQ ID NO: 11 (h139Ab-hCD28Z) specifically lysescell lines engineered to express the mutant EGFRvIII.

The ability of EGFRvIII CAR engineered T cells to lyse target cells in astandard ⁵¹Cr-release assay was next determined (FIGS. 1A-D and 2A-D).

Untransduced (UnTd) PBL or PBL transduced with control GFP vector (GFP),139-28Z CAR (vIII-28Z) (encoding SEQ ID NO: 11), or 139-28BBZ (vIII-BBZ)(encoding SEQ ID NO: 10) were co-cultured for four hours with ⁵¹Crlabeled target tumor cell lines (FIGS. 1A-1D: parent U87, GFP, wild typeEGFR, or EGFRvIII engineered).

Untransduced (UnTd) PBL or PBL transduced with control GFP vector (GFP),anti-ERBB2 CAR (ERBB2), 139-28Z CAR (vIII-28Z) (encoding SEQ ID NO: 11)or 139-28BBZ (vIII-BBZ) (encoding SEQ ID NO: 10) were co-cultured forfour hours with ⁵¹Cr labeled target tumor cell lines (FIGS. 2A-2D:parent U251, GFP, wild type EGFR, or EGFRvIII engineered).

In the experiments of FIGS. 1A-1D and 2A-2D, specific lysis of tumorcells was measured at the given E:T ratio using the formula: [(specificrelease-spontaneous release)/total release-spontaneous release)]. Asshown in FIGS. 1A-1D and 2A-D, both vectors specifically lysed only celllines engineered to express the mutant EGFRvIII and not control orwild-type EGFR engineered cell lines.

EXAMPLE 3

This example demonstrates that an anti-EGFRvIII CAR (SEQ ID NO: 10(h139Ab-hCD828BBZ)) produces IFN-gamma following co-culture with tumorstem cell (TSC) lines.

By detailed molecular analysis of many different classes of cancer celllines, it has now been demonstrated that established cancer cell linesoften do not mirror the molecular characteristics of primary humancancers and this is the case for glioma lines. An alternative to the useof established glioma cell lines is the analysis of tumor stem cell(TSC) lines. The TSC paradigm proposes that a subpopulation of cellsexist in cancer that give rise to all the cells in a differentiatedtumor. It has been demonstrated that in situ glioma cells shareproperties not found in glioma cell lines, and harbor featuresconsistent with tumor stem cells. It was further demonstrated thatmarked phenotypic and genotypic differences exist between primary humantumor-derived TSCs and their matched glioma cell lines. TSCs deriveddirectly from primary glioblastomas harbor extensive similarities tonormal neural stem cells and recapitulate the genotype, gene expressionpatterns, and in vivo biology of human glioblastomas. These findingssuggest that glioma-derived TSCs may be a more reliable model than manycommonly utilized glioma cell lines for understanding the biology ofprimary human tumors.

Therefore three TSC lines were analyzed for the presence of EGFRvIII anddemonstrated by RT-PCR that EGFRvIII is expressed in these lines. PBLfrom two donors (Effector I and Effector II) were then engineered withthe anti-EGFRvIII CAR vector (expressing SEQ ID NO: 10(h139Ab-hCD828BBZ)) and co-cultured with glioma TSC lines and controlEGFRvIII expressing cell lines. Five post-transduction PBL wereco-cultured with glioma TSC lines or cell line U251 that had beenengineered to express wild type EGFR, or EGFRvIII. Untransduced (UT)cells and GFP transduced cells served as negative controls and ananti-ERBB2 CAR served as a positive control in all co-cultures. As shownin FIGS. 5A and 5B, EGFRvIII CAR engineered T cells demonstratedspecific recognition of the U251 EGFRvIII, when compared to the U251EGFR wild type gene-engineered cells, and recognized all three gliomaTSC lines tested (308, 822, and 1228). These results further support theuse of EGFRvIII CAR engineered T cells as a potential immunotherapy forglioma patients.

EXAMPLE 4

This example demonstrates that CAR-engineered T-cells retain reactivityfollowing expansion of the number of T-cells.

The 139-28BBZ (h139Ab-hCD828BBZ) vector was used to transduce PBL fromtwo glioblastoma patients, as well as a healthy donor and tested forexpression and reactivity. Transduced cells were co-cultured withEGFRvIII-engineered U87 cells and then assayed by intracellular cytokinestaining. Engineered T cells from the patients and the healthy donordemonstrated specific IFN-γ production in both CD8+ and CD8− (presumablyCD4+) CD3+ T cells (7.8%-16.2% IFN-γ+, vs. >0.36% against the controlU87 line). Transduction efficiency was also similar between theglioblastoma patient T cells and the healthy donor. If large numbers ofT cells (>1×10⁹) are required for future clinical applications, thesecan be obtained via, for example, a 14-day rapid expansion protocol(REP) (Riddell et al., J. Immunol. Methods, 128: 189-201 (1990)). Toverify that 139-28BBZ (h139Ab-hCD828BBZ) CAR transduced T cells could beexpanded to numbers sufficient for patient treatment, and still maintainreactivity, these T cells were subject to REP and retested. The 139-CARtransduced T cells retained their ability to specifically produce IFN-γas shown in Table 4.

TABLE 4 IFN-γ Elispot per 1 × 10⁶ cells Donor 6 GBM-1 GBM-2 UT CAR UTCAR UT CAR U87 0 <500 0 0 0 <500 vIII 0 >5500 0 >5500 0 >5500 PHA 50003500 2800 1500 4500 5000

EXAMPLE 5

This example demonstrates the production of a producer cell clone usefulfor producing viral vector supernatant for transducing cells.

Using the 139-28BBZ (h139Ab-hCD828BBZ) EGFRvIII CAR construct, a PG13γ-retroviral vector producer cell clone was produced under conditionsthat meet U.S. Food and Drug Administration (FDA) guidelines for humangene therapy clinical trials. One cell clone (clone F10) was used toproduce 18 L of viral vector supernatant in 6 harvests collected over 4days. Each harvest was used to transduce donor PBL and the gene transferefficiency and biologic activity were determined. All harvests producedbiologically active supernatant based on the ability of transduced Tcells to express the CAR and to specifically recognize EGFRvIIIexpressing cell lines. Harvest 1 was slightly less reactive than harvest2-6 in this assay. To test for possible toxicity against normal humantissues, a pool of harvests 3 and 4 was used to transduce a differentdonor and these transduced T cells were co-cultured with seven differentprimary human adult and neonatal-cell cultures of epithelial,endothelial, and fibroblast origin. As determined by IFN-γ production,there was no reactivity of the EGFRvIII CAR transduced T cells with anyprimary human cell culture tested.

EXAMPLE 6

This example demonstrates a method of treating or preventing cancer in ahuman patient comprising administering to the patient a CAR comprisingSEQ ID NO: 10 (h139Ab-hCD828BBZ).

Eligibility

Eligible patients have histologically proven glioblastoma expressingEGFRvIII as determined by immunohistochemistry (IHC); failed priorstandard treatment with radiotherapy with or without chemotherapy; aKarnofsky score greater than or equal to 60%; cardiac, pulmonary, andlaboratory parameters within acceptable limits.

Study Design

The study is conducted using a Phase I/II design. Patients are accruedto both the Phase I and Phase II portion of the trial in two groups: 1)patients with recurrent malignant glioma requiring steroid use at thestart of treatment or 2) patients with recurrent malignant glioma notrequiring steroid use at the start of treatment. Once the maximumtolerated dose is determined for each individual group in the phase Iportion of the trial, the study proceeds to the phase II portion.Patients are again accrued to the same two groups. For each of the twogroups evaluated, the study is conducted using a single stage phase IIdesign.

Patients receive a non-myeloablative but lymphocyte depletingpreparative regimen including cyclophosphamide and fludarabine followedby intravenous infusion of ex vivo tumor reactive, EGFRvIII CARgene-transduced PBMC, plus intravenous (IV) aldesleukin (720,000 IU/kgq8h for a maximum of 15 doses). Patients undergo complete evaluation oftumor with physical and neurological examination, MRI of the brain withand without gadolinium, and clinical laboratory evaluation four weeks(+/−7 days) after completion of treatment. If the patient has stabledisease or tumor shrinkage, repeat complete evaluations are performedevery 1 month (+/−7 days. After the first year, patients continuing torespond continue to be followed with this evaluation every 2 months(+/−7 days) as appropriate.

Cell Preparation

PBMC are obtained by leukapheresis (approximately 1×10¹⁰ cells). WholePBMC are cultured in the presence of anti-CD3 (OKT3) and aldesleukin inorder to stimulate T-cell growth. Transduction is initiated by exposureof approximately 1'10⁷ to 5×10⁸ cells to supernatant containing theanti-EGFRvIII CAR retroviral vector. These transduced cells are expandedand tested for their anti-tumor activity. Successful CAR gene transferis determined by FACS analysis for the CAR protein and anti-tumorreactivity is tested by cytokine release as measured on EGFRvIIIexpressing cells. Successful CAR gene transfer for each transduced PBLpopulation is defined as >10% CAR positive cells and for biologicalactivity, gamma-interferon secretion must be at least 200 pg/ml andtwice the background level.

Anti-EGVRvIII CAR Transduced PBL

The PBL are transduced with retroviral supernatant containing thechimeric anti-EGFRvIII CAR. The retroviral vector supernatant(PG13-139-F10) encoding a chimeric antigen receptor (CAR) directedagainst the antigen, EGFRvIII, is prepared and preserved followingcurrent good manufacturing practice (cGMP) conditions. The retroviralvector utilizes the MSGV1 retroviral vector backbone and includes 4,032bps including the 5′ LTR from the murine stem cell virus (promoter),packaging signal including the splicing donor (SD) and splicing acceptorsites, human anti-EGFRvIII scFv-based (mAb 139) CAR protein containing asignal peptide signal (human GM-CSFR), 139 light chain variable region,linker peptide, 139 heavy chain variable region, CD8 (hinge,transmembrane), CD28 (cytoplasmic region), 4-1BB (cytoplasmic region)and TCR zeta (cytoplasmic region), followed by the murine stem cellvirus 3′LTR. The vector comprises nucleotide sequence SEQ ID NO: 13,which encodes amino acid sequence SEQ ID NO: 10. The physical titer isdetermined by RNA dot blot according to sponsor certificate. Thesupernate is stored at SBVPF upon the completion of production at −80°C. with around-the-clock temperature monitoring. Upon request,supernatant is delivered on dry ice to be used in in vitro transduction.There is no re-use of the same unit of supernate for different patients.Retroviral titer has been shown to be stable after immediate thawing andimmediate administration (coating the tissue culture wells previouslycoated with Retronectin). Handling of the vector follows the guidelinesof Biosafety Level-2 (BSL-2).

Phase I—Dose Escalation

The protocol begins with a phase 1 dose escalation design, with eightcohorts and with two different groups (one for patients receivingsteroids at the time of treatment and one for patients not on steroids).Each group is treated as a totally separate dose escalation trial.

Initially, the protocol enrolls 1 patient in each of the first 3 dosecohorts unless that patient experiences a dose limiting toxicity (DLT).Following cohort 3, all subsequent cohorts proceed in a phase 1 doseescalation design, with 5 cohorts of n=3.

The total number of EGFRvIII engineered cells transferred for eachcohort is according to Table 5:

TABLE 5 Dose Escalation Schedule Dose Level Dose of Anti-EGFRvIII CARTcells Cohort 1 (group a & b) 10⁷ Cohort 2 (group a & b) 3 × 10⁷ Cohort 3(group a & b) 10⁸ Cohort 4 (group a & b) 3 × 10⁸ Cohort 5 (group a & b)10⁹ Cohort 6 (group a & b) 3 × 10⁹ Cohort 7 (group a & b)  10¹⁰ Cohort 8(group a & b) 3-6 × 10¹⁰

Patients are enrolled sequentially, therefore enrollment does notproceed to a higher dose level until patients have been treated in theprior cohort. Patients, however, are dose-escalated to the next cohortwithin a given group independent of what is occurring in the otherstrata. If sufficient cells cannot be grown to meet the criteria for theassigned cohort, the patient is enrolled in the appropriate cohort forthe number of cells infused.

In cohorts 1 through 3, if the patient experiences a DLT, five morepatients would be treated at that dose to confirm that no greater than ⅙patients have a DLT prior to proceeding to the next higher level. If alevel with 2 or more DLTs in 3-6 patients has been identified, fiveadditional patients are accrued at the next-lowest dose, for a total of6, in order to further characterize the safety of the maximum tolerateddose prior to starting the phase II portion. If there are 1 or fewerDLTs in the first cohort, the study proceeds to the second cohort. If adose limiting toxicity occurs in the first cohort, that cohort isexpanded to n=6 patients. If two DLTs occur in the first cohort, thestudy is terminated.

In cohorts 4-8, should a single patient experience a dose limitingtoxicity due to the cell infusion at a particular dose level, three morepatients would be treated at that dose to confirm that no greater than ⅙patients have a DLT prior to proceeding to the next higher level. If alevel with 2 or more DLTs in 3-6 patients has been identified, threeadditional patients are accrued at the next-lowest dose, for a total of6, in order to further characterize the safety of the maximum tolerateddose prior to starting the phase II portion.

The maximum tolerated cell dose is the highest dose at which <1 of 6patients experienced a DLT or the highest dose level studied if DLTs arenot observed at any of the three dose levels.

Prior to receiving the engineered PBL cells, all patients receive anonmyeloablative, but lymphocyte depleting preparative regimen,including cyclophosphamide and fludarabine followed in one to four daysby intravenous infusion of in vitro tumor reactive, EGFRvIII CARgene-transduced PBL plus IV aldesleukin (720,000 IU/kg q8h for a maximumof 15 doses).

The maximum tolerated cell dose is the highest dose at which <1 of 6patients experienced a DLT or the highest dose level studied if DLTs arenot observed at any of the three dose levels.

Dose-limiting toxicity is defined as follows: Grade 2 or greaterallergic reaction or reaction that involves bronchospasm or generalizedurticaria; all grade 3 and 4 toxicities with the exception of:myelosuppression, defined as lymphopenia, neutropenia andthrombocytopenia; IL-2 expected toxicities; toxicities occurring within24 hours post cell infusion (related to cell infusion) that arereversible to a grade 2 or less within 8 hours with two doses ofacetaminophen (650 mg) or two doses of diphenhydramine (25 mg).

Treatment Schedule

The treatment schedule is set forth in Table 6:

TABLE 6 Day Therapy −7 −6 −5 −4 −3 −2 −1 01 1 2 3 4 Cyclophosphamide (60mg/kg) X X Fludarabine (25 mg/m²) X X X X X Anti-EGFRvIII CAR PBL X¹Aldesleukin X² X X X X Filgrastim³ (5 mcg/kg/day) X X X X trimethoprimand X X X X X X X X  X X X X sulfamethoxazole (TMP/SMX)⁴ 160 mg/800 mgFluconazole⁵ (400 mg po) X  X X X X Valacyclovir po or Acyclovir IV⁶ X X X X X ¹One to four days after the last dose of fludarabine ²Initiatewithin 24 hours after cell infusion ³Continue until neutrophils count >1× 10⁹/L for 3 consecutive days or >5 × 10⁹/L. ⁴The TMP/SMX scheduleshould be adjusted to QD three times per week (Monday, Wednesday,Friday) and continue for at least six months and until CD4 >200 × 2⁵Continue until ANC >1000/mm³ ⁶In patients positive for HSV continueuntil ANC is greater than 1000/mm³

Immunological Testing

Apheresis is performed prior to, and 4-6 weeks after, the treatment. Atother time points, patient peripheral blood lymphocytes (PBL) areobtained from whole blood by purification using centrifugation on aFicoll cushion. Aliquots of these PBMC are 1) cryopreserved forimmunological monitoring of cell function, 2) subjected to DNA and RNAextraction for PCR analysis of CAR and vector copy number estimation,and 3) lymphocytes are tested directly and following in vitro culture.Direct immunological monitoring includes quantifying T cells reactivewith EGFRvIII by FACS analysis using CAR-specific staining. Ex vivoimmunological assays include cytokine release by bulk PBL (+/−antigenstimulation) and by other experimental studies such as cytolysis ifsufficient cells are available. If cell numbers are limiting, preferenceis given to the direct analysis of immunological activity. Immunologicalassays are standardized by the inclusion of 1) pre-infusion PBMC and 2)an aliquot of the engineered PBL cryopreserved at the time of infusion.In general, differences of 2 to 3 fold in these assays are indicative oftrue biologic differences.

Monitoring Gene Therapy Trials: Persistence and Replication-CompetentRetrovirus (RCR)

Engineered cell survival: CAR and vector presence is quantitated in PBMCsamples using established PCR techniques. Immunological monitoring usingCAR-specific staining is used to augment PCR-based analysis. Thisprovides data to estimate the in vivo survival of lymphocytes derivedfrom the infused cells. In addition, measurement of CD4 and CD8 T-cellsis conducted and studies of these T-cell subsets in the circulation aredetermined by using specific PCR assays capable of detecting the uniqueDNA sequence for each retroviral vector engineered T-cell.

Patients' blood samples are obtained and undergo analysis for detectionof RCR by PCR prior to cell infusion and RCR PCR is performed at 3 and 6months, and at one year post cell administration. Blood samples arearchived annually thereafter if all previous testing has been negativewith a brief clinical history. If a patient dies or develops neoplasmsduring this trial, efforts are made to assay a biopsy sample for RCR. Ifany post-treatment samples are positive, further analysis of the RCR andmore extensive patient follow-up is undertaken, in consultation with theFDA. RCR PCR assays detect the GaLV envelop gene and are performed undercontract by the National Gene Vector Laboratory at Indiana UniversityThe results of these tests are maintained by the contractor performingthe RCR tests and by the National Cancer Institute (NCI) Surgery Branchresearch team.

Due to the nature of these studies, it is possible that expansion ofspecific T-cell clones is observed as tumor reactive T-cellproliferation in response to tumor antigens. Therefore, care is taken totrack T-cell persistence both immunologically and molecularly. Bloodsamples (5-10 mL) for persistence of CAR transduced cells are obtained 1month after cell infusion, then at 3, 6, 12 months, and then annuallythereafter. If any patient shows a high level of persistence of CAR genetransduced cells at month 6 (by semi quantitative DNA-PCR using primersspecific for vector sequences) the previously archived samples aresubjected to techniques that would allow the identification of clonalityof persisting CAR gene transduced cells. Such techniques may include Tcell cloning or LAM-PCR 30. If a predominant or monoclonal T cell clonederived from CAR gene transduced cells is identified during thefollow-up, the integration site and sequence are identified andsubsequently analyzed against human genome database to determine whetherthe sequences are associated with any known human cancers. If apredominant integration site is observed, the T cell cloning or LAM-PCRtest is used at an interval of no more than three months after the firstobservation to see if the clone persists or is transient. In allinstances where monoclonality is persistent and particularly ininstances where there is expansion of the clone, regardless of whetheror not the sequence is known to be associated with a known human cancer,the subject should be monitored closely for signs of malignancy, so thattreatment, if available, may be initiated early.

Post Treatment Evaluation (Follow-Up)

Routine Follow up: Patients are evaluated 4 weeks (+/−7 days) after theinitial treatment regimen (defined as the end of the last aldesleukindose). If the patient has SD or tumor shrinkage, repeat completeevaluations are performed monthly (+/−7 days) for 12 months, and thenevery 1-2 months (+/−7 days) as appropriate.

The following evaluations are performed at each evaluation: I) Physicalexamination, including neurological examination and Karnofsky score; II)Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin,Calcium total, Magnesium total (Mg), Inorganic Phosphorus, AlkalinePhosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD,Total Protein, Total CK, Uric Acid), complete blood count and thyroidpanel; III) CBC; IV) Toxicity assessment; V) MRI of the brain with andwithout gadolinium; and VI) Detection of RCR and persistence of CAR genetransduced cells: (as described above).

A 5 liter apheresis is performed at the first follow up visit only.Subsequently, 60 ml of blood is obtained at follow up visits(approximately monthly) for at least 3 months. Peripheral bloodmononuclear cells are cryopreserved so that immunologic testing may beperformed.

Long-Term Follow Up of Patients Receiving Gene Transfer

Physical examinations are performed and documented annually for 5 yearsfollowing cell infusion to evaluate long-term safety. After 5 years,health status data are obtained from surviving patients via telephonecontact or mailed questionnaires. The long term follow up period forretroviral vectors is 15 years.

Response Criteria

As part of this trial, as well as to assist in the determination oftumor progression, all efforts are made to observe radiographic changesin the patient's tumors over time.

Measurable Disease: Bidimensionally contrast-enhancing lesions withclearly defined margins by MRI scan, with two perpendicular diameters ofat least 10 mm, visible on two or more axial slices. Measurement oftumor around a cyst or surgical cavity represents a particularlydifficult challenge. In general, such lesions should be considerednonmeasurable unless there is a nodular component measuring ≥10 mm indiameter. The cystic or surgical cavity should not be measured indetermining response.

Non-Measurable but Evaluable Disease: Unidimensionally measurablelesions, masses with margins not clearly defined, or lesions with amultiple cystic component.

Non-Evaluable Disease: No definitive, measurable or evaluable tumor.

Measurable Lesions

Complete Response (CR): Complete response requires all of the following:complete disappearance of all enhancing measurable and nonmeasurabledisease sustained for at least 4 weeks; no new lesions; stable orimproved nonenhancing (T2/FLAIR) lesions; and patient must be offcorticosteroids or on physiologic replacement doses only, and stable orimproved clinically. In the absence of a confirming scan 4 weeks later,this response is considered only stable disease.

Partial Response (PR): Partial response requires all of the following:≥50% decrease, compared with baseline, in the sum of products ofperpendicular diameters of all measurable enhancing lesions sustainedfor at least 4 weeks; no progression of nonmeasurable disease; no newlesions; stable or improved nonenhancing (T2/FLAIR) lesions on same orlower dose of corticosteroids compared with baseline scan; and patientmust be on a corticosteroid dose not greater than the dose at time ofbaseline scan and is stable or improved clinically. In the absence of aconfirming scan 4 weeks later, this response is considered only stabledisease.

Stable: Stable disease occurs if the patient does not qualify forcomplete response, partial response, or progression and requires thefollowing: stable nonenhancing (T2/FLAIR) lesions on same or lower doseof corticosteroids compared with baseline scan and clinically stablestatus. In the event that the corticosteroid dose was increased for newsymptoms and signs without confirmation of disease progression onneuroimaging, and subsequent follow-up imaging shows that this increasein corticosteroids was required because of disease progression, the lastscan considered to show stable disease is the scan obtained when thecorticosteroid dose was equivalent to the baseline dose.

Progression: Progression is defined by any of the following: ≥25%increase in sum of the products of perpendicular diameters of enhancinglesions (compared to best response or with baseline if no decrease) onstable or increasing doses of corticosteroids; a significant increase inT2/FLAIR nonenhancing lesions on stable or increasing doses ofcorticosteroids compared with baseline scan or best response afterinitiation of therapy, not due to comorbid events; the appearance of anynew lesions; clear progression of nonmeasurable lesions; or definiteclinical deterioration not attributable to other causes apart from thetumor, or to decrease in corticosteroid dose. Failure to return forevaluation as a result of death or deteriorating condition should alsobe considered as progression. Patients with nonmeasurable enhancingdisease whose lesions have significantly increased in size and becomemeasurable (minimal bidirectional diameter of ≥10 mm and visible on atleast two axial slices) are also be considered to have experiencedprogression. The transition from a nonmeasurable lesion to a measurablelesion resulting in progression can theoretically occur with relativelysmall increases in tumor size (eg, a 9×9 mm lesion [nonmeasurable]increasing to a 10×11 mm lesion [measurable]). Ideally, the changeshould be significant (>5 mm increase in maximal diameter or ≥25%increase in sum of the products of perpendicular diameters of enhancinglesions). In general, if there is doubt about whether the lesion hasprogressed, continued treatment and close follow-up evaluation helpclarify whether there is true progression.

Evaluable Lesions

Evaluable lesions are recorded at each evaluation. FLAIR or T2-weightedimages should also be assessed as evaluable disease if appropriate.

The following scale is used to designate relative changes in MRI scans:

-   -   +3=disappearance of tumor (CR)    -   +2=definitely better (PR)    -   +1=possibly better    -   0=unchanged    -   −1=possibly worse    -   −2=definitely worse (PD)    -   −3=development of a new lesion (PD).

Definition of Response for Evaluable Lesions

Complete Response (CR): is defined as the circumstance when the MRI scanis ranked +3 and the tumor is no longer seen by neuroimaging, and thepatient no longer requires steroids for control of tumor-inducedcerebral edema.

Partial response (PR): is defined as a MRI scan ranked +2 provided thatthe patient has not had his/her dose of steroids increased since thelast evaluation period.

Progression (P): is defined as the circumstance when the MRI scan isranked −2 or −3, or the presence of a new lesion.

Stable disease (SD): is defined as the circumstance when the MRI scanshows no change or possible (−1 or +1) changes. Patients should bereceiving stable or decreasing doses of steroids.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of treating cancer in a mammal,the method comprising administering to the mammal a population of cellsin an amount effective to treat cancer in the mammal, wherein thepopulation of cells comprises one or more T cells expressing a chimericantigen receptor (CAR) having antigen specificity for epidermal growthfactor receptor variant III (EGFRvIII), wherein the CAR comprises anamino acid sequence selected from the group consisting of SEQ ID NOS:10-11, wherein the cancer is an EGFRvIII positive cancer, and whereinthe population of cells is autologous to the mammal.
 2. The methodaccording to claim 1, wherein the cancer is glioma.
 3. The methodaccording to claim 1, wherein the cancer is ependymoma, astrocytoma,oligodendroglioma, oligoastrocytoma, or glioblastoma multiforme (GBM).4. The method according to claim 1, wherein the population of cells is apopulation of peripheral blood lymphocytes.
 5. The method according toclaim 1, wherein the population of cells is a population of peripheralblood mononuclear cells.
 6. The method according to claim 1, wherein thepopulation of cells is a heterogeneous population of cells.
 7. Themethod according to claim 1, wherein the population of cells is ahomogeneous population of cells.
 8. A method of treating cancer in amammal, the method comprising administering to the mammal a populationof cells in an amount effective to treat cancer in the mammal, whereinthe population of cells comprises one or more T cells expressing achimeric antigen receptor (CAR) having antigen specificity for epidermalgrowth factor receptor variant III (EGFRvIII), wherein the CAR comprisesan amino acid sequence selected from the group consisting of SEQ ID NOS:10-11, wherein the cancer is an EGFRvIII positive cancer, and whereinthe population of cells is allogeneic to the mammal.
 9. The methodaccording to claim 8, wherein the cancer is glioma.
 10. The methodaccording to claim 8, wherein the cancer is ependymoma, astrocytoma,oligodendroglioma, oligoastrocytoma, or glioblastoma multiforme (GBM).11. The method according to claim 8, wherein the population of cells isa population of peripheral blood lymphocytes.
 12. The method accordingto claim 8, wherein the population of cells is a population ofperipheral blood mononuclear cells.
 13. The method according to claim 8,wherein the population of cells is a heterogeneous population of cells.14. The method according to claim 8, wherein the population of cells isa homogeneous population of cells.